以FKBP51为靶点的小分子抑制前列腺癌AR信号通路

发布时间：2018-03-31 11:16

本文选题：前列腺癌　切入点：FKBP51　出处：《兰州大学》2017年博士论文

【摘要】：目的:前列腺癌(PCa)是男性癌症最常见的恶性肿瘤之一。对于晚期患者除了手术治疗以外,常常使用抗雄药物作为辅助治疗。这些抗雄药物多是靶向于雄激素受体(AR),竞争性地与雄激素结合在活性位点,阻碍AR信号通路并且抑制前列腺癌细胞的生长,但这些药物的治疗效果却未令人满意。随着结构生物学的发展,肿瘤的药物开发越来越多依赖关键靶蛋白的结构,成为肿瘤药物开发的主要方法。有研究报道在前列腺癌细胞中,FKBP51是AR的正调控因子,通过抑制FKBP51的活性能够有效抑制AR的转录活性,降低AR的信号通路。本课题以FKBP51为主要靶点,通过虚拟方法筛选方法,筛选出小分子抑制剂,抑制PCa细胞的生长。同时,前人报道小分子-对硝基酚(环境内分泌干扰物)具有抗雄活性,它的抗雄机制是复杂的,可能涉及了AR信号通路多个成分,但它是怎样抑制AR活性的并不知道。另外,雷帕霉素(rapamycin)是FKBP51已知的小分子抑制剂,但它对AR信号通路的影响是未知的,因此,我们研究是否它对AR信号通路的作用,以及是否通过FKBP51蛋白介导。本实验将FKBP51作为筛药的靶向蛋白和分子抗雄作用的关键蛋白,旨在研究FKBP51在前列腺癌信号通路中的作用,并进一步探讨小分子药物或者污染物通过FKBP51作用的可能的抗雄机制,为PCa及CRPC的治疗提供一种新的治疗策略,尤其对于雄激素抵抗型前列腺癌的药物设计及临床治疗提供了一定的理论基础和实验依据。方法:计算机虚拟筛选靶向FKBP51的小分子抑制剂,根据打分选择前140名的虚拟筛选小分子抑制剂;构建FK1-PET-28a质粒,通过大肠杆菌诱导表达并纯化FKBP51的FK1区域;通过蛋白质荧光淬灭实验-酶标法高通量检测蛋白质与虚拟筛选小分子的相互作用,并用荧光滴定法测结合较好的小分子的结合常数。不同温度下通过蛋白质荧光淬灭实验-分光光度法检测小分子(对硝基酚、rapamycin)与FKBP51的相互作用;将小分子与蛋白质共结晶,通过分子结构学方法X-晶体衍射分析小分子与蛋白质结合复合物的结构,分析它们结合的特征等;通过分子动力学方法检测分子小分子与蛋白结合的复合物的稳定性;采用MTT比色法检测小分子对前列腺细胞生长与增殖的影响;利用qRT-PCR检测小分子对AR所调控的下游基因mRNA水平的影响;通过Western blot检测小分子对AR、FKBP51与PSA蛋白的表达水平的影响。结果:(1)诱导和纯化FK1区域,纯化后鉴定纯度超过99%。(2)对硝基酚(PNP)能够与FKBP51的FK1区域相互作用,随着PNP滴定浓度的增加,蛋白的荧光强度减弱;随着温度的增加,它们的结合增强,在人体生理温度下(310k)它们的结合力最强。(3)X-晶体衍射分析FK1与PNP结合的复合物的结构,PNP占据了FK1区域的活性位点,与未结合小分子的蛋白相比,复合物结构更加坚固。(4)分子动力学揭示了FK1与PNP的结合非常稳定。(5)计算FK1区域与PNP的结合自由能得出小分子与蛋白主要通过非极性键相互作用。(6)计算与PNP结合的FK1区域的每个热点氨基酸的结合自由能,显示出与X-晶体衍射结构分析一致的热点氨基酸。(7)不同浓度的PNP在48小时抑制了前列腺癌LNCaP和22Rv1细胞的生长与增殖。(8)PNP抑制了AR所调控的下游基因(PSA,KLK2,S100P,TMPRSS2)的mRNA水平的表达,从而抑制了AR信号通路。(9)计算机虚拟筛选得到FKBP51小分子抑制剂,前140名能够与FKBP51蛋白相互作用。(10)得到结合较强的前10个虚拟筛选小分子的解离常数,其中四个小分子的Kd值小于20μM。(11)虚拟筛选小分子能够抑制LNCaP细胞的生长和增殖。(12)不同浓度的Rapamycin在24小时和48小时抑制了前列腺癌LNCaP细胞和22rv1细胞生长。(13)Rapamycin能够与FKBP51相互作用。(14)X-晶体衍射分析FK1与rapamycin结合的复合物的结构,rapamycin占据了FK1区域的活性口袋结构。(14)Rapamycin能够抑制AR调控的下游基因mRNA的表达。(5)Rapamycin抑制了AR调控的下游基因PSA蛋白水平的表达。结论:本实验发现了PNP能够与FKBP51蛋白结合及PNP在前列腺癌细胞的抗雄机理,即PNP抑制AR的信号通路可能通过与FKBP51相互作用,抑制其活性进而抑制了AR的活性,降低AR下游基因的表达,抑制前列腺癌细胞的生长。另外,我们运用虚拟筛选方法,以FKBP51为靶点,得到了一些有效抑制前列腺癌细胞生长的小分子,并且在分子水平上验证了与FKBP51的相互作用。同时,作为FKBP51的抑制剂rapamycin,我们通过X-晶体衍射揭示了rapamycin与FKBP51的结合方式,同时证明了rapamycin能够降低AR的信号通路,抑制前列腺癌细胞的生长。
[Abstract]:Objective: prostate cancer (PCa) is one of the most common malignant tumor in male cancer. For patients with advanced surgical treatment in addition to outside, often using anti drugs as adjuvant therapy. These male male anti drug is targeting androgen receptor (AR), competing with androgen binding in the active site, and inhibition of prostate cancer cell block AR signaling in growth, but the therapeutic effect of these drugs are not satisfactory. With the development of structural biology, drug development and tumor structure more dependent critical target protein, has become the main method of tumor drug development. Studies have reported in prostate cancer cells, FKBP51 is a positive regulator of AR transcription. The activity by inhibiting the activity of FKBP51 can effectively inhibit AR, reduce the AR signaling pathway. This paper takes FKBP51 as the main target, through the method of virtual screening method, screening of small molecule inhibitors, Inhibit the growth of PCa cells. At the same time, previous reports of small molecule - nitrophenol (EDCs) with anti male activity, its anti male mechanism is complex and may involve multiple components of the AR pathway, but it is how to inhibit the activity of AR does not know. In addition, Lei rapamycin (rapamycin) is a small molecule inhibitor FKBP51 is known, but its effect on the AR signaling pathway is unknown, therefore, we investigated whether its effect on the AR signaling pathway, and whether it is mediated by FKBP51 protein. The key protein FKBP51 as a drug screening target anti male role to proteins and molecules, to effect study of FKBP51 signaling pathway in prostate cancer, and further explore the small molecule drugs or contaminants through the mechanism of anti male possible role of FKBP51, and provides a new therapeutic strategy for the treatment of PCa and CRPC, especially for male steroid resistance To provide a theoretical basis and experimental basis for prostate cancer drug design and clinical treatment. Methods: virtual screening targeted small molecule inhibitors of FKBP51, according to the choice of scoring virtual screening of small molecule inhibitors of the top 140; the construction of FK1-PET-28a plasmid, FK1 region by Escherichia coli expression and purification of FKBP51 by protein interaction; fluorescence quenching experiments - ELISA high-throughput protein detection and virtual screening of small molecules, and fluorescence titration with the binding constants of small molecule better. Under different temperatures through the protein fluorescence quenching experiment - spectrophotometric detection of small molecules (rapamycin p-nitrophenol,) the interaction with FKBP51 the small molecules and proteins; CO crystallization by molecular structure method of X- crystal diffraction analysis of small molecules and protein structure with complex analysis, they combine the characteristics of The stability of the complexes; by combining molecular detection method of molecular dynamics of small molecules and proteins; by MTT colorimetric detection of small molecule effects on growth and proliferation of prostate cells; effect of downstream gene mRNA level using the qRT-PCR detection of small molecular regulation of AR; through Western blot detection of small molecules on AR expression the level of FKBP51 and PSA protein. Results: (1) induction and purification of the FK1 region, after purification. The purity of more than 99%. (2) p-nitrophenol (PNP) can interact with the FKBP51 FK1 region, with the increase of PNP titration concentration, the fluorescence intensity of the protein decreased; with the increase of temperature, enhance their in combination, human physiological temperature (310K) combined with the strongest of them. (3) X- crystal diffraction analysis of FK1 and PNP combined with the structure of the complexes, PNP occupy the active sites in FK1 region, compared with the combination of small molecule protein composite The structure is more solid. (4) molecular dynamics reveals the combination of FK1 and PNP are very stable. (5) can be obtained free of small molecules and protein mainly through non polar bond interaction with the calculated FK1 region and PNP. (6) binding free energy of each hot spot calculation of FK1 and PNP combined with the amino acid region of the show, and X- crystal diffraction structure analysis of hot amino acids identical. (7) different concentrations of PNP inhibited the growth and proliferation of LNCaP cells and 22Rv1 prostate cancer in 48 hours. (8) PNP inhibits downstream genes regulated by AR (PSA, KLK2, S100P, TMPRSS2) expression level of mRNA. To inhibit the pathway of AR. (9) computer virtual screening by small molecule inhibitors of FKBP51, to the top 140 and FKBP51 protein interaction. (10) obtained with dissociation constants of 10 before the virtual screening of small molecule strong, including four small molecular Kd value of less than 20 M. (11) deficiency The growth and proliferation of quasi screening of small molecules could inhibit LNCaP cells. (12) different concentrations of Rapamycin in 24 hours and 48 hours inhibits prostate cancer LNCaP cells and 22Rv1 cells. (13) Rapamycin can interact with FKBP51. (14) X- crystal diffraction analysis of FK1 and rapamycin combined with the structure of the complex rapamycin occupied the active pocket structure of the FK1 region. (14) the expression of downstream genes mRNA Rapamycin can inhibit the regulation of AR. (5) Rapamycin inhibited the expression of downstream genes PSA protein level in the regulation of AR. Conclusion: This study found that PNP can combine with FKBP51 protein and PNP in the mechanism of anti male prostate cancer cells that is, the inhibition of PNP signaling pathway of AR may interact with FKBP51 and inhibit its activity and inhibit the activity of AR, reduce the expression of downstream genes of AR, inhibit the growth of prostate cancer cells. In addition, we use virtual screening Methods FKBP51 as a target to get some effective to inhibit the growth of prostate cancer cells of small molecules, and verify the interaction with FKBP51 at the molecular level. At the same time, as the FKBP51 inhibitor rapamycin, we reveal the combination of rapamycin and FKBP51 by X- crystal diffraction, and proves that rapamycin can reduce the signal pathway AR, inhibit the growth of prostate cancer cells.

【学位授予单位】：兰州大学
【学位级别】：博士
【学位授予年份】：2017
【分类号】：R737.25

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